1. Area of the Art
The present invention relates to an improved method for producing increased amounts of safe coagulation factor concentrates from blood plasma
2. Description of the Prior Art
There are a number of medical indications for administration of xe2x80x9cclottingxe2x80x9d or xe2x80x9ccoagulationxe2x80x9d factors from human blood. These factors are proteins that cause the clotting of blood to staunch bleeding from wounds, etc. Individuals with any of a series of genetic abnormalities affecting the proteins responsible for blood coagulation are afflicted with a disease (hemophilia) in which the blood fails to clot normally, subjecting the individual to the danger of uncontrolled bleeding. For many years this condition has been treated by administering concentrates of the missing or defective proteins. Many clotting factors are synthesized in the liver so that victims of liver disease are also in need of augmentation of their clotting factors. Additionally, there are other important medical uses for clotting-related factors.
While some of the clotting factors are currently produced through biotechnology, at this time there is still no cost effective method of artificially manufacturing all of these proteins. Further, the xe2x80x9cartificially producedxe2x80x9d factors tend to be more expensive. Many of the xe2x80x9cminorxe2x80x9d factors are not yet (and may never be) available from biotechnology sources and so must be purified from donated human blood. This is especially true in Third World countries where the biotechnology products are generally not affordable. Therefore, much of the supply of antihemophilia factor (AHF, also known as Factor VIII), and other blood clotting factors are prepared from pooled human plasma. A hemophiliac requires treatment for a whole lifetime. Victims of liver disease and other users of clotting factors may also require prolonged treatment. Therefore, these patients are exposed to blood products produced from the blood of a large number of donors.
The presence of AIDS (Acquired Immuno Deficiency Syndrome) virus or HIV in the blood supply means that hemophiliacs and other users of clotting factors have become infected with this terrible disease. Although tests to screen out AIDS-tainted blood have been improved, some infected blood does slip by. Even if the AIDS problem is solved, the danger of other blood-borne diseases, such as the various types of hepatitis and other, as yet unknown, infectious agents, makes it desirable to reduce or eliminate virus and other disease organisms from plasma used to prepare clotting factors. One way of achieving this goal is to reduce the use of pooled plasma products since xe2x80x9cone bad apple spoils the entire barrelxe2x80x9d. However, even with the use of clotting factors derived from a single donor, there is still danger. Even though tests may show the donor is free of known disease, the donor may be incubating a disease that will later show on the tests or the donor may harbor a yet unknown disease or a yet unknown strain of a known disease. These dangers have been lessened by use of plasma pre-treatments that inactivate disease organisms. Unfortunately, the available treatments either do not inactivate all types of disease organisms or damage the labile clotting factors during the process of inactivating disease organisms.
The basic methods for preparing clotting factor concentrates from blood have not changed greatly over the last few decades. Generally, a concentrate of clotting factors is derived from pooled plasma by a cryoprecipitation step. Various additives such as ethanol or polyethylene glycol are usually added to enhance the efficiency of the cryoprecipitation step. Following cryoprecipitation, the partially purified factors may be further purified by additional precipitation steps or by chromatographic methods, and most recently by methods utilizing monoclonal antibodies. For additional information on the basic techniques of clotting factor purification and the history of the development of these methods, the reader is directed to U.S. Pat. Nos. 3,560,475. 3,631,018, 3,682,881, 4,069,216, and 4,305,871 and 5,770,704 by the present inventor, the contents of which are incorporated herein by reference, and the references cited therein.
It is an object of the present invention to provide enhanced yields of cryoprecipitate;
It is a further object of the present invention to inactivate and/or enhance the inactivation of disease organisms within plasma at the same time that cryoprecipitate production is enhanced.
Derivatives of simple carboxylic acids, particularly trisodium citrate and other citrate salts (hereinafter xe2x80x9ccitratexe2x80x9d) are shown to be unexpectedly effective agents for enhancing the production of blood clotting factors. It is believed that other small carboxylic acids may show similar properties. Isocitric acid, in particular, appears to show these properties. To date most of the tests have been made with citric acid and its salts. Addition of citrate to plasma, especially at concentrations between 2 and 10% by weight, does not appreciably denature labile proteins. However, in this concentration range citrate is effective in inactivating or inhibiting a variety of pathogenic microorganisms. Further, the added citrate potentiates or enhances the killing of microorganisms by heat treatment. That is, heating of the material to relatively low temperatures (i.e., above 45xc2x0 C.) which do not denature proteins enhances the killing of microorganisms in the presence of citrate. Significantly, added citrate causes a dramatic increase in the weight of cryoprecipitate that can be produced from plasma by the usual procedures. The majority of significant clotting factors are greatly concentrated in the resulting cryoprecipitate. The supernatant contains little if any of these clotting factors. It is apparent that increasing the amount of citrate in blood bags so that the final concentration will be at least 2% by weight results in plasma that can be used to produce improved platelet concentrates and enhanced cryoprecipitate. The added citrate can help eliminate or suppress contaminating microorganisms and can itself be removed by ion exchange or similar methods well known in the art.